Diabetes Volume 64, February 2015 573 Dieter Rondas,1 Inne Crèvecoeur,1 Wannes D’Hertog,1 Gabriela Bomfim Ferreira,1 An Staes,2,3 Abhishek D. Garg,4 Decio L. Eizirik,5 Patrizia Agostinis,4 Kris Gevaert,2,3 Lut Overbergh,1 and Chantal Mathieu1 Citrullinated Glucose- Regulated Protein 78 Is an Autoantigen in Type 1 Diabetes Diabetes 2015;64:573–586 | DOI: 10.2337/db14-0621 Posttranslational modifications of self-proteins play a sub- the autoimmune assault progresses, with autoantibodies – fi stantial role in the initiation or propagation of the autoim- appearing against several non b-cell-speci c autoantigens, IMMUNOLOGY AND TRANSPLANTATION mune attack in several autoimmune diseases, but their such as GAD65 (3), islet antigen 2 (IA2) (4), heat shock contribution to type 1 diabetesisonlyrecentlyemerging.In protein 60 (HSP60) (5), and chromogranin A (ChgA) (6). the current study, we demonstrate that inflammatory stress, During insulitis, local production of inflammatory media- induced by the cytokines interleukin-1b and interferon-g, tors, such as the cytokines interleukin (IL)-1b and interferon-g leads to citrullination of GRP78 in b-cells. This is coupled (IFNg), triggers b-cell oxidative and endoplasmic reticulum with translocation of this endoplasmic reticulum chaperone (ER) stress. These, and other signals, may lead to alternative to the b-cell plasma membrane and subsequent secretion. splicing and misfolding of b-cellproteinsaswellasposttrans- Importantly, expression and activity of peptidylarginine lational modifications (PTMs) (7–9).Inotherautoimmune deiminase 2, one of the five enzymes responsible for citrul- diseases, like rheumatoid arthritis (RA), multiple sclerosis, lination and a candidate gene for type 1 diabetes in mice, is and celiac disease, such posttranslationally modified proteins increased in islets from diabetes-prone nonobese diabetic behave as autoantigens (10,11), but their relevance in type 1 (NOD) mice. Finally, (pre)diabetic NOD mice have autoanti- – bodies and effector T cells that react against citrullinated diabetes is only starting to be explored (12 15). GRP78, indicating that inflammation-induced citrullination Building on our previous observation that the ER of GRP78 in b-cells generates a novel autoantigen in type 1 chaperone 78 kDa glucose-regulated protein (GRP78; also diabetes, opening new avenues for biomarker development named binding immunoglobulin protein [BiP]) is post- and therapeutic intervention. translationally modified in cytokine-exposed insulin- producing INS-1E cells (9), we now identify this modification as citrullination and show that inflammation-induced Type 1 diabetes is an autoimmune endocrine disease in citrullinated GRP78 is an autoantigen in type 1 diabetes. which loss of central and peripheral tolerance toward b-cell These findings suggest a novel role for GRP78 beyond its antigens is proposed as the underlying mechanism. How- well-known function in the ER, leading to the loss of ever, b-cells themselves also contribute to trigger and/or tolerance to b-cells in type 1 diabetes. propagate the autoimmune attack, leading to a dialogue with immune infiltrating cells that may amplify local in- RESEARCH DESIGN AND METHODS flammation (insulitis) in genetically predisposed individuals Western blotting, mass spectrometry, GRP78 cloning, (1). Insulin (or proinsulin) is probably the primary autoan- expression, purification, and in vitro citrullination are tigen in type 1 diabetes (2), but antigen spreading occurs as available in the Supplementary Data. 1Laboratory for Clinical and Experimental Endocrinology, KU Leuven, Leuven, This article contains Supplementary Data online at http://diabetes Belgium .diabetesjournals.org/lookup/suppl/doi:10.2337/db14-0621/-/DC1. 2 Department of Medical Protein Research, VIB, Ghent, Belgium D.R. and I.C. contributed equally to this study. 3Department of Biochemistry, Ghent University, Ghent, Belgium L.O. and C.M. contributed equally to this study. 4Laboratory for Cell Death Research and Therapy, KU Leuven, Leuven, Belgium 5Laboratory of Experimental Medicine and Université Libre de Bruxelles Center for © 2015 by the American Diabetes Association. Readers may use this article as Diabetes Research, Medical Faculty, Université Libre de Bruxelles, Brussels, Belgium long as the work is properly cited, the use is educational and not for profit, and the work is not altered. Corresponding author: Lut Overbergh, [email protected]. Received 18 April 2014 and accepted 28 August 2014. 574 GRP78 Is an Autoantigen in Type 1 Diabetes Diabetes Volume 64, February 2015 Reagents and Antibodies Islet Isolation and Culture Primary antibodies were as follows: mouse anti-poly(ADP- Pancreatic islets were isolated from 3- or 10-week-old ribose) monoclonal antibody (mAb) (Enzo Life Sciences, C57Bl/6, NOD, and NOR mice. Islet isolation and culture Antwerp, Belgium), rabbit anti-GRP78 and anti-CHOP were performed as previously described (16). C57Bl/6 polyclonal antibody (pAb) (Santa Cruz Biotechnology, islets were exposed to recombinant mouse IFNg (1,000 Santa Cruz, CA), rabbit anti-eIF2a pAb, anti–p-eIF2a units/mL) and recombinant human IL-1b (50 units/mL). (Ser51) pAb, anti-PERK mAb and anti–p-PERK(Thr980) Immunofluorescence mAb (Cell Signaling, Beverly, MA), and mouse anti-actin Immunofluorescence on isolated islets was performed as mAb (Sigma-Aldrich, Diegem, Belgium) for Western blot- ting; rabbit anti–b-catenin mAb (Cell Signaling Technol- previously described (17). Fixed INS-1E cells or sectioned islets were incubated with primary antibody in 1% BSA ogy) and mouse anti-GRP78 pAb (Abcam, Cambridge, for 1 h, followed by four washes in PBS before incubation U.K.) for immunocytochemistry. Anti–cit(510)-GRP78 with the secondary antibody in 1% BSA for another hour. was raised in rabbits against the following peptides: C- Nuclei were detected with DNA-binding dye DRAQ5TM aminohexanoic acid-IDVNGIL[citrulline]VTAEDKG-amide (Biostatus Ltd., Leicestershire, U.K.). Specificity was con- and acetyl-IDVNGIL[citrulline]VTAEDKG-aminohexanoic firmed by including negative controls with secondary anti- acid-C-amide, through five subsequent injections (21st fi bodies alone. INS-1E samples were observed under a Zeiss Century Biochemicals). Speci city of the antibody was fl 3 confirmed by dot blot against the citrullinated peptide LSM 510 microscope using a Plan-Neo uar 40 /1.3 oil DIC lens. Images were acquired and processed using LSM and its native counterpart. Secondary antibodies were as 510 software (Carl Zeiss AG, Jena, Germany). Mouse islet follows: donkey anti-rabbit horseradish peroxidase, don- sections were observed under a Nikon Eclipse Ti micro- key anti-mouse Alexa Fluor 488, and donkey anti-rabbit scope using a Plan-Fluor 403/0.75 DIC lens, and images Alexa Fluor 555 (Invitrogen, Merelbeke, Belgium). Oval- were acquired and processed using Nis-Elements Viewer bumin and rabbit peptidylarginine deiminase (PAD) en- 4.20 software (Nikon Instruments Inc.). zyme were from Sigma-Aldrich. Cell Surface Biotinylation Cell Lines and Culture Conditions INS-1E or MIN6 cells were incubated with the indicated Rat INS-1E cells, a gift from Prof. Wollheim (CMU, stressors for 12–15 h and then treated as previously de- Geneva, Switzerland), were cultured as previously de- scribed (18). scribed (9). INS-1E cells were exposed to recombinant rat IFNg (500 units/mL; R&D Systems), recombinant GRP78 ELISA human IL-1b (10 units/mL; R&D Systems), thapsigargin To determine GRP78 concentration in conditioned media (Tg) (15 and 50 nmol/L; Sigma-Aldrich), tunicamycin from INS-1E cells, the GRP78 ELISA kit (Enzo Life (Tn) (2 and 5 mg/mL; Sigma-Aldrich), high glucose Sciences, Antwerp, Belgium) was used, according to the (HG) (25 mmol/L; Sigma-Aldrich), and palmitate (Pa) manufacturer’s protocol. (0.5 mmol/L; Sigma-Aldrich). Mouse MIN6 cells, a gift Two-Dimensional Gel Electrophoresis Analysis from Dr. Miyazaki (Osaka University, Osaka, Japan), Two-dimensional gel electrophoresis (2D-GE) analysis was were cultured in DMEM (Invitrogen) containing 15% performed as previously described (9). (volume for volume) FCS, 100 units/mL penicillin, 100 mg/mL streptomycin, and 70 mmol/L b-mercaptoethanol. Quantitative RT-PCR MIN6 cells were exposed to recombinant mouse IFNg (500 Quantitative RT-PCR was performed as previously de- units/mL; R&D Systems) and human recombinant IL-1b scribed (19). (10 units/mL). Measurement of PAD Activity Apoptosis Measurements To determine PAD activity levels in islets and pancreata The percentage of living and apoptotic cells was assessed from C57Bl/6, NOR, and NOD mice, the antibody-based as previously described (9). assay for PAD activity (ABAP) (ModiQuest Research, Oss, the Netherlands) was used, according to the manufac- Mice turer’s protocol. C57Bl/6 mice were obtained from Harlan Laboratories (Horst, the Netherlands) and nonobese resistant (NOR) Autoantibody ELISA Against Native or Citrullinated mice from The Jackson Laboratory (Bar Harbor, ME). GRP78 Nonobese diabetic (NOD) mice have been inbred in our Serum autoantibodies against two native and citrullinated animal facility since 1989 and are kept under semi- GRP78 peptides (amino acids 500–519 TFEIDVNGILRV barrier conditions. For all experiments, a mix of male and TAEDKGTG and amino acids 295–314 AKRALSSQHQARI female mice was used. All animal manipulations were EIESFYE) were determined by ELISA as previously de- in compliance with the principles of laboratory care and scribed (20). For the citrullinated forms, arg510 or arg306 approved